We are currently experiencing an unprecedented times in biological/biomedical sciences with enumerate new, rapid, sensitive and high throughput technologies to study protein-structure function. One of these technologies is state-of-the-art mass spectrometry which has the capacity to characterize more globally the proteomes in body fluids. Saliva has the potential to reflect a measure of health/disease state, since it contains a wide range of proteins, analytes, antibodies and hormones derived from blood as well as those specific to glandular secretions. The identification of the "phosphoproteome of saliva" requires the use of combination of specific chemistries and tools applicable to phosphopeptide biochemistry. Phosphoproteomics to date is an untouched area in the saliva proteomics field, despite the fact that it is the phosphoprotein contents of saliva that have been demonstrated to be of vital importance in the protection of tooth enamel. We hypothesize that there are major differences in the phosphoproteome of oral fluids derived from whole saliva, major and minor glandular secretions and gingival crevicular fluid (GCF). It is both highly relevant and timely to define the differential phosphoproteome of each of these contributors to the general oral fluid since results of such work will most likely lead to the identification of novel unknown salivary phosphoproteins, and find direct applications in clinical dentistry. Our recent work with whole saliva from healthy individuals led to identification of a large-scale phosphoproteome of this fluid with 114 phosphoproteins identified of which ~95% were hitherto unknown. We further hypothesize that the phosphoproteome of whole saliva differs significantly from those of glandular salivary secretions and GCF. Indeed, our most recent study established the parotid saliva phosphoproteome which identified 40 phosphoproteins, only ~1/3 that of the whole saliva phosphoproteome. These studies for the first time culminated in providing direct evidence in support of our conceptual developments and hypothesis. This is consistent with our hypothesis that glandular phosphoproteome can be modified in whole saliva by e.g., phosphatases, or that additional phosphoproteome contributions are in effect, e.g., GCF. We expect that GCF will have its own distinct phosphoproteome due to its close proximity to the bone which contains abundant extracellular matrix phosphoproteins. The presence of bone phosphoproteins in GCF is eminent during bone resorption by osteoclasts in periodontal disease. We will investigate the phosphoproteome of GCF samples from subjects with normal oral health and those with periodontal disease. The proposed studies will develop and implement efficient approaches to the phosphoproteome analysis of oral fluids by combining our expertise in phosphoprotein chemistry/biochemistry interfaced with the latest state-of-the-art mass spectrometric technology. The accomplishments of the proposed work herein will contribute significantly and add another dimension to the future development of saliva based diagnostics/biomarkers and new therapeutics with considerable health and economic impact. PUBLIC HEALTH RELEVANCE: Phosphoproteins fulfill an essential role in the maintenance of the integrity of teeth. With the advances in mass spectrometric techniques, a global spectrum of phosphoproteins and their distribution amongst the salivary glands can now be established. Such knowledge will be invaluable in determining the full protective potential of phosphoproteins in an oral environment and help to develop novel therapeutic approaches for the treatment of oral and/or systemic human diseases.